title: Promoter characterization and role of CRE in the basal transcription of the rat SNAT2 gene.
abstract: Small neutral amino acid transporter 2 (SNAT2) is the most abundant and ubiquitous transporter for zwitterionic short-chain amino acids. The activity of this amino acid transporter is stimulated in vivo or in vitro by glucagon or cAMP analogs. However, it is not known whether the increase in activity at the protein level is due to an increase in SNAT2 gene transcription. Thus, the aim of the present work was to study whether cAMP was able to stimulate SNAT2 gene expression and to localize and characterize the presence of cAMP response elements (CRE) in the promoter that controls the expression of the rat SNAT2 gene. We found that consumption of a high-protein diet that increased serum glucagon concentration or the administration of glucagon or incubation of hepatocytes with forskolin increased the SNAT2 mRNA level. We then isolated the 5' regulatory region of the SNAT2 gene and determined that the transcriptional start site was located 970 bp upstream of the translation start codon. We identified two potential CRE sites located at -354 and -48 bp. Our results, using deletion analysis of the 5' regulatory region of the SNAT2 gene, revealed that the CRE site located at -48 bp was fully responsible for SNAT2 regulation by cAMP. This evidence was strongly supported by mutation of the CRE site and EMSA and ChIP analysis. Alignment of rat, mouse, and human sequences revealed that this CRE site is highly conserved among species, indicating its essential role in the regulation of SNAT2 gene expression.
authors.affiliation: Departamento de Fisiología de la Nutrición, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga 15, Mexico.
authors.forename: Victor
authors.initials: V
authors.lastname: Ortiz
authors.forename: Gabriela
authors.initials: G
authors.lastname: Alemán
authors.forename: Martín
authors.initials: M
authors.lastname: Escamilla-Del-Arenal
authors.forename: Félix
authors.initials: F
authors.lastname: Recillas-Targa
authors.forename: Nimbe
authors.initials: N
authors.lastname: Torres
authors.forename: Armando R
authors.initials: AR
authors.lastname: Tovar
mesh_terms: D026924:Amino Acid Transport System A; D026905:Amino Acid Transport Systems / Q000096:biosynthesis* / Q000235:genetics*; D000818:Animals; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D003001:Cloning, Molecular; D003198:Computer Simulation; D000242:Cyclic AMP / Q000502:physiology; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology*; D004032:Diet; D004044:Dietary Proteins / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000235:genetics / Q000502:physiology; D005934:Glucagon / Q000097:blood / Q000494:pharmacology; D005943:Gluconeogenesis / Q000235:genetics; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006706:Homeostasis / Q000502:physiology; D006801:Humans; D048088:Informatics; D008297:Male; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D012313:RNA; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013045:Species Specificity; D015854:Up-Regulation
chemical_list: D026924:Amino Acid Transport System A; D026905:Amino Acid Transport Systems; D017362:Cyclic AMP Response Element-Binding Protein; D004044:Dietary Proteins; C034477:RNA primers; C412568:Slc38a2 protein, rat; D012313:RNA; D005934:Glucagon; D000242:Cyclic AMP